In the treatment of diabetes mellitus insulin preparations derived from porcine or bovine insulin have generally been used. Bovine, porcine, and human insulins exhibit minor differences with respect to their amino acid sequence, the difference between human and porcine insulin being confined to a single amino acid in that the B30 amino acid of human insulin is threonine whereas that of porcine insulin is alanine. However, it could be argued that the ideal insulin preparation for human beings would be an insulin having exactly the same chemical structure as that of human insulin.
For the production of natural human insulin the necessary amount of human pancreas glands is not available.
Synthetic human insulin has been prepared on a small scale at great expense, vide Helv. Chim. Acta 57, 2617, and 60, 27.
Semisynthetic human insulin has been prepared from porcine insulin by what are believed to be tedious pathways, vide Hoppe-Zeyler's A. Physiol. Chem. 356, 1631, and Nature 280, 412.
One known semisynthetic process for preparing human insulin comprises the following three steps: First, porcine insulin is converted into porcine des-(Ala.sup.B30)-insulin by treatment with carboxypeptidase A, vide Hoppe-Zeyler's A. Physiol. Chem. 359, 799. In the second step porcine des-(Ala.sup.B30)-insulin is subjected to a trypsin-catalyzed coupling with Thr-OBu.sup.t, whereby human insulin Thr.sup.B30 -tert-butyl ester is formed. Finally, said ester is treated with trifluoroacetic acid yielding human insulin, vide Nature 280, 412. The first step, however, results in a partial removal of Asn.sup.A21, yielding des-(Ala.sup.B30, Asn.sup.A21)-insulin. This derivative gives, after the two subsequent reactions, rise to a contamination by des-(Asn.sup.A21)-insulin in the semisynthetic human insulin product, a contamination which cannot easily be removed with known preparative methods. Des-(Asn.sup.A21)-insulin possesses low biological activity (about 5%), vide Amer. J. Med. 40, 750.
U.S. Pat. No. 3,276,961 purports to relate to a transpeptidation process for preparing semisynthetic human insulin directly from porcine insulin. However, the yield of human insulin is poor because the process is performed in water, under which conditions trypsin causes splitting of the Arg.sup.B22 -Gly.sup.B23 bond, vide J. Biol. Chem. 236, 743.
The invention is based upon the discovery that the amino acid or peptide chain bound to the carbonyl group of Lys.sup.B29 in the insulin compound can be interchanged with a threonine ester. Said interchange is herein referred to as a transpeptidation.
The term "insulin compounds" as used herein encompasses insulins and insulin-like compounds containing the human des(Thr.sup.B30) insulin moiety, the B30 amino acid of the insulin being alanine (in insulin from, e.g., hog, dog, and fin and sperm whale) or serine (rabbit). The term "insulin-like compounds" as used herein encompasses proinsulin derived from any of the above species and primates, together with intermediates from the conversion of proinsulin into insulin. As examples of such intermediates can be mentioned split proinsulin, desdipeptide propinsulins, desnonapeptide proinsulin, and diarginine insulins, vide R. Chance: In Proceedings of the Seventh Congress of IDF, Buenos Aires 1970, 292-305, Editors: R. R. Rodriques & J. V.-Owen, Excerpta Medica, Amsterdam.
One object of this invention is to provide a process for converting certain non-human insulins and insulin-like compounds into a threonine B30 ester of human insulin in high yields.
A second object of this invention is to provide a process for converting crude porcine insulin into a threonine B30 ester of human insulin in high yields.